Chapter30

Chapter30 - BCH 4054 Chapter 30 Lecture Notes Slide 1...

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Chapter 30, page 1 BCH 4054 Chapter 30 Lecture Notes Slide 1 Chapter 30 DNA Replication and Repair Slide 2 Conceptual Mechanism of Replication • Strand separation, with copying of each strand by Watson-Crick base pairing • Fig 30.2 • Three models for replication • Conservative • Semiconservative • Dispersive • See Fig 30.3 Slide 3 Meselson Stahl Experiment • DNA labeled with 15 NH 4 + • Culture diluted with excess 14 NH 4 + • DNA isolated and analyzed by ultracentrifugation (See Fig 30.4) • Original DNA bands at 1.724 g/mL • First generation bands at 1.717 g/mL • Second generation bands at 1.710 g/mL These density labeling experiments are most consistent with the semiconservative mechanism of replication. Subsequent analysis of the single strands of the hybrid after the first generation ruled out the dispersive mechanism.
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Chapter 30, page 2 Slide 4 Problems with Semiconservative Model • Strands run in opposite directions • Twisted strands must come apart • Error rate must be very low • (< 1 per 10 9 -10 10 bases copied) Slide 5 Chemical Components of Replication • Template • DNA strand determining order of new bases • Primer • Strand grows at 3’ end • Deoxynucleotide triphosphates • “monomer” unit is activated, adding to free 3’- OH of the primer • See Fig 30.8 The alternative would be for the terminal end of the primer to carry the triphosphate group, with linkage made to OH of the monomer unit. Slide 6 Some Features of Replication • Bidirectional • Two replication “forks” which move in opposite direction. • Fig 30.5 • Semidiscontinuous • “Leading” strand copied continuously • “Lagging” strand made in segments (Okasaki fragments) which must be joined. • Fig 30.6
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Chapter 30, page 3 Slide 7 Enzymology: DNA Polymerase • Three different polymerases in E. coli • Pol I, Pol II, and Pol III (order of discovery) • See Table 30.1 • Pol I—monomer of 103 kD • 400 molecules/cell; turnover 600/min • Pol II—monomer of 90 kD • ? Molecules/cell; turnover 30/min • Pol III—complex (“core” contains 3 subunits) • 40 molecules/cell; turnover 1200/min Pol III, the third disovered, does the bulk of new DNA synthesis. Pol I has a role in normal replication, but is also involved in DNA repair. Pol II is probably involved only in repair. Slide 8 DNA Polymerase I • First discovered • Arthur Kornberg, 1957 • Catalyzes incorporation of radioactive nucleotides into polymeric form • Requires primer, template, and all dNTP’s • Two additional enzymatic activities • 3’-5’ exonuclease • 5’-3’ exonuclease Slide 9 Klenow Fragment of Pol I • Contains the 5’-3’ polymerase and 3’-5’ exonuclease activity Fig 30.10 diagram shows protein-DNA interaction and two enzymatic sites • Fig 30.9 shows model of fragment • 3’-5’ Exonuclease has proofreading function
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Chapter30 - BCH 4054 Chapter 30 Lecture Notes Slide 1...

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